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Nucleotide-induced global conformational changes of flagellar dynein arms revealed by in situ analysis

Nature Structural & Molecular Biology volume 17, pages 761–767 (2010)Cite this article

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Abstract

Outer and inner dynein arms generate force for the flagellar/ciliary bending motion. Although nucleotide-induced structural change of dynein heavy chains (the ATP-driven motor) was proven in vitro, our lack of knowledge in situ has precluded an understanding of the bending mechanism. Here we reveal nucleotide-induced global structural changes of the outer and inner dynein arms of Chlamydomonas reinhardtii flagella in situ using electron cryotomography. The ATPase domains of the dynein heavy chains move toward the distal end, and the N-terminal tail bends sharply during product release. This motion could drive the adjacent microtubule to cause a sliding motion. In contrast to in vitro results, in the presence of nucleotides, outer dynein arms coexist as clusters of apo or nucleotide-bound forms in situ. This implies a cooperative switching, which may be related to the mechanism of bending.

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Figure 1: Schematic diagram of a flagellum and dynein arms.
Figure 2: ODA 3D structure of in situ in the presence and absence of nucleotides.
Figure 3: Surface rendering representations of IDAs and ODAs as well as other components on A-microtubules averaged from tomograms using 96-nm periodicity.
Figure 4: Structure of ODAs revealed by electron cryotomography and image averaging based on 24-nm periodicity.
Figure 5: The orientation and conformation of the N-terminal tails and stalks of ODA.
Figure 6: Linear arrangement of ODAs along microtubules in the presence of ADP.Vi as revealed by 3D image classifications.

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Acknowledgements

We thank T.J. Richmond for the biochemical facility, D. Sargent and F. Damberger for critical reading of the manuscript, R. Wepf, P. Tittmann and F. Stribel for technical support, J.A. Parian for support on graphics, V.H. Bui for statistical calculation and S. Burgess for insightful discussion. This work was funded by grants from the Swiss National Science Foundation (NF3100A0-107540 and NF31003A-125131/1), a Swiss-Japan cooperative grant and NCCR Structural Biology to T.I., by Special Coordination Funds for Promoting Science and Technology (16083207 to K.O.) and a Grant-in-Aid for Scientific Research on the Priority Area “Regulation of Nano-systems in Cells” by the Japan Ministry of Education, Culture, Sports, Science and Technology (K.O.).

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Author notes

  1. Tandis Movassagh and Khanh Huy Bui: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, ETH Zurich, Zurich, Switzerland

    Tandis Movassagh, Khanh Huy Bui & Takashi Ishikawa

  2. Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Iwaoka, Kobe, Japan

    Hitoshi Sakakibara & Kazuhiro Oiwa

  3. Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo, Japan

    Kazuhiro Oiwa

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  1. Tandis Movassagh
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Contributions

T.I. designed experiments; T.M. and H.S. prepared specimens and performed biochemical experiments; T.M. collected tomography data; K.H.B. developed algorithms of image alignment and classification and made programs; T.M. and K.H.B. performed image analysis of ODAs and IDAs, respectively; the mechanism of the power stroke was discussed by all the authors; the manuscript was written by T.I., T.M., K.O. and K.H.B.

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Correspondence to Takashi Ishikawa.

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The authors declare no competing financial interests.

Supplementary information

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Supplementary Methods and Supplementary Figures 1–7 (PDF 968 kb)

Supplementary Video 1

Fitting a ring model to the density of the inner arm dynein c at various threshold levels and seen from different view angles (MOV 3126 kb)

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Movassagh, T., Bui, K., Sakakibara, H. et al. Nucleotide-induced global conformational changes of flagellar dynein arms revealed by in situ analysis. Nat Struct Mol Biol 17, 761–767 (2010). https://doi.org/10.1038/nsmb.1832

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